Cross-talk between kinases and proteases in tumour necrosis factor-#alpha# receptor subtype-induced apoptosis
Firstly, we demonstrated that caspase-dependent cell death was enhanced by the over-expression of the type II TNF receptor (TNFR2). HeLa cells, which naturally express high levels of type I TNF receptor (TNFR1) and low levels of TNFR2, were engineered to stably over-express TNFR2. This combined with the use of genetically-engineered mutated TNFs that preferentially activate TNFR1 or TNFR2, we showed that both receptors can induce cell death, although this process occurred predominantly through TNFR1. TNF-induced cell death was inhibited by the stable expression of cytokine response modifier A (CrmA), a potent inhibitor of receptor proximal caspases. By isolating early apoptotic cells, we were able to identify differential activation profiles of members of the mitogen-activated protein kinase (MAPK) family during TNF-induced apoptosis. In dying HeLa-TNFR2 cells, there was increased activation of c-Jun NH2-terminal kinase (JNK) while the activation levels of p38 MAPK and p42/44 MAPK remained unchanged. The use of peptidergic caspase inhibitors demonstrated that caspase-dependent modulation of JNK but not p38 MAPK or p42/44 MAPK takes place, and as such may provide a mechanism which accounts for the differences observed in MAPK activity during TNF-induced cell death. Through expression of a dominant negative upstream activator of JNK (SEK-1-AL) and a pharmacological inhibitor of JNK activity, we were able to determine the role of JNK activation in TNF receptor-mediated apoptosis. These findings clearly demonstrate that through cross-talk, TNF receptors, are able to further modulate the tightly regulate cellular consequences of TNF treatment and that JNK is a TNF-induced kinase that may play a role in the cytokine’s apoptotic cellular signalling.